br Introduction Identification and correct storage of semina
Introduction Identification and correct storage of seminal stains are crucial for the investigation of sexual assault cases. Actually, undetected ‘cold’ cases involving sexual assault can be solved decades after investigations analysing DNA from stored evidences . Recently, STR analysis and the successful typing rate in 19 extremely old seminal stain were assessed . In February 2015, during the restoration work of the museum of the Institute of Legal Medicine at the University of Bologna, three pieces of cotton fabric were found inside an old envelope of the Institute showing handwritten “seminal stains”. It was estimated that the seminal stains date back to the first half of the ‘900, when the institute had been transferred in the today’s site and Baecchi’s staining was already used also for student’s training. The aim of this study was to evaluate the forensic implication of the analysis on the three aged seminal stains using current forensic methods including the semenogelin test, microscopic identification of spermatozoa, autosomal, Y-chromosome and X chromosome STRs, and mitochondrial DNA analysis.
Materials and methods The three pieces of cotton fabric were submitted to the analysis: evidence no. 1 was a white handkerchief with visible yellow stiff stains and lacerations perhaps related to multiple cuts for old sampling; evidence no. 2 was a piece of white and green cotton fabric showing brown shade and stiff brown stains; evidence no. 3 was a piece of an yellowed handkerchief with visible brownish stains. Specimens of presumed seminal stains were collected from each evidence by sterile blades and were submitted to semen-specific semenogelin test using RSID Semen laboratory kit (Independent Forensics, USA) according to the manufacturer’s protocol with an incubation time of 2h. Christmas tree staining was performed on the pellet of 1cm×1cm specimens of stains, previously incubated in sterile water o.n. at room temperature and prior of DNA extraction by Qiagen Investigator kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Quantification of extracted DNA was performed by 7500 Real-Time PCR System (ThermoFisher Scientific, Oyster Point, CA) using the commercial Quantifiler Trio DNA quantification Kit (ThermoFisher Scientific, Oyster Point, CA). STRs analysis was performed by PowerPlex® ESX 17 Fast System (Promega, Madison, USA), AmpFlSTR Minifiler PCR XL-888 Kit (ThermoFisher Scientific, Oyster Point, CA), PowerPlex® Y23 System (Promega, Madison, USA), and Investigator Argus X-12 QS Kit (Qiagen, Hilden, Germany). RM-Y STRs was analysed according to Robino et al. . Mitochondrial DNA analysis was performed for HVI region as described in Bini et al. . For evidences no. 2 and no. 3 the analysis were performed in double on two specimens each. STRs and mt-DNA detection were performed by ABI 310 Genetic Analyzer (Thermo Fisher Scientific, Oyster Point, CA) followed for STRs by GeneMapper® ID v3.2 software analysis (ThermoFisher Scientific, Oyster Point, CA).
Results All samples displayed a weak positive reaction for semenogelin test and the sperm heads were clearly identified in all the samples, always pooled in groups perhaps due to the difficult extraction from the substrates. In Table 1 the DNA quantity with relative degradation index and the number of loci detected by STRs analysis are summarized. For evidence no. 3b the reported full profiles were composite profiles obtained by two PCR reaction with different amount of target DNA. HVI mt-DNA was successfully amplified only for evidence no. 1.
Discussion DNA degradation index for the analyzed specimens was strongly correlated (r=−0.95) with the number of detectable autosomal STRs amplified by PowerPlex® ESX 17 Fast System and with number of detected Y-STRs (r=−0.97) if evidence no. 3b results were not included in r calculation. Actually, the full profiles of this specimen were obtained increasing in PCR reaction the quantity of DNA target up to 2ng. This strategy resolved allelic imbalance and possible allelic dropout for homozygous loci in larger STRs observed when lower DNA quantity were amplified, but numerous artifacts for smaller STRs were obtained as reported , interfering with the correct peaks designation. For evidence no. 2, the quantities of recovered DNA were low, but full profiles for the 9 STRs included in the Minifiler kit and for smaller STRs in ESX System were reproducible in all analysed specimens. Therefore, for old seminal stains, lacking the reference DNA samples and considering that the possibility of allelic dropout for homozygous loci can interfere with the correct typing, the selection of amplicons size based on degradation index should be preliminary to casework analysis and composite profiles strategy could be advisable when, despite the high degradation index, enough DNA target is available. The same results were obtained also for Y-chromosome and the new RM Y-STRs analysis, suitable in rape casework also for predicting population of origin and for differentiation of paternally related males, but multicopy loci of RM Y-STR resulted particularly affected by DNA degradation. X-chromosome STRs analysis, useful also for paternity testing when child is female, was successfully performed also for evidence no. 2, for which 9/12 loci were typed. The correlation between DNA degradation index and the number of X-STR amplifiable was strong (r=−0.97). HVI Mitochondrial DNA amplification results suggest that typing should be performed by shorter amplicon yet described  according to the degradation index.